This proposal is concerned with the development of an in vitro nucleolar system to be used for the identification and characterization of ribonucleases involved in ribosomal RNA processing. Nucleoli will be prepared from Ehrlich ascites carcinoma cells. The 80S ribonucleoprotein particle of the 45S pre-ribosomal RNA will be used as the substrate in processing studies. RNA products from the ribonuclease processing of the 80S RNP particle will be separated by electrophoresis on agarose gels. Hybridization with ribosomal DNA probes containing either 18S or 28S ribosomal RNA sequences will be used to determine the sequences represented in RNA products. The enzymatic properties of a ribonuclease identified in this investigation will be studied in relationship to the constraints placed upon its action by its own specificity, by substrate structure and modification, and by the nucleolar environment. An in vitro nucleolar transcriptional system which mimics the in vivo situation will be used to elucidate the effect of nucleolar ribonuclease action directly on transcription.